Biliary tract cancer organoid derived from human chemically-derived hepatic progenitors (hcdhs) using crispr-cas9

Abstract

The present disclosure relates to a biliary tract cancer organoid and a method of producing the same. A new biliary tract cancer organoid system was developed in which CRISPR-Cas9 gene editing technology is applied to human chemically-derived hepatic progenitors to introduce mutations into some genes known as tumor suppressors or oncogenes, and the effects of the combined genetic mutations on human non-tumor, chemically-derived hepatic progenitors are analyzed to identify and screen the effects of the combined mutations on oncogenesis.

What is claimed is: 
     
         1 . A method of producing a biliary tract cancer organoid, comprising:
 a step of culturing human primary hepatocytes (hPHs) in a reprogramming medium containing hepatocyte growth factor (HGF), A83-01, and CHIR99021 as reprogramming factors to reprogram the hPHs into human chemically-derived hepatic progenitors (hCdHs);   a step of reprogramming by inducing loss of function of TP53 and BAP1 genes of hCdHs based on a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) system containing a CRISPR-Cas9 guide RNA (gRNA); and   a step of subculturing the hCdHs reprogrammed in the above step in a reprogramming medium containing HGF, CHIR99021, and A83-01 to form a biliary tract cancer organoid.   
     
     
         2 . The method of  claim 1 , wherein the HGF is contained in the reprogramming medium at a concentration of 2 to 100 ng/ml. 
     
     
         3 . The method of  claim 1 , wherein the A83-01 is contained in the reprogramming medium at a concentration of 0.4 to 4 μM. 
     
     
         4 . The method of  claim 1 , wherein the CHIR99021 is contained in the reprogramming medium at a concentration of 0.3 to 3 μM. 
     
     
         5 . The method of  claim 1 , wherein the hPHs are cultured for 3 to 14 days. 
     
     
         6 . The method of  claim 1 , wherein the reprogramming medium is one or more selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal essential medium (MEM), Basal medium eagle (BME), RPMI1640, F-10, F-12, α-Minimal essential medium (α-MEM), Glasgow's Minimal essential medium (GMEM), and Iscove's Modified Dulbecco's Medium (IMDM). 
     
     
         7 . The method of  claim 1 , wherein the CRISPR-Cas9 guide RNA for the TP53 gene is one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. 
     
     
         8 . The method of  claim 1 , wherein the CRISPR-Cas9 guide RNA for the BAP1 gene is one or more selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:
 6, SEQ ID NO: 7, and SEQ ID NO: 8.   
     
     
         9 . The method of  claim 1 , wherein the subculture is performed by culturing human hepatic progenitors in a reprogramming medium for one to three passages and mixing the human hepatic progenitors and the reprogramming medium in a volume ratio of 1:3 to 1:5 every five days. 
     
     
         10 . A biliary tract cancer organoid produced according to the method of  claim 1 , capable of establishing a biliary tract cancer-inducing environment with a short production period and limited samples. 
     
     
         11 . A method of screening for the prognosis of biliary tract cancer, comprising:
 a step of bringing a test substance into contact with the biliary tract cancer organoid of claim  10 ; and   a step of measuring cell viability in the biliary tract cancer organoid.   
     
     
         12 . A method of screening drugs for ameliorating or treating biliary tract cancer, comprising:
 a step of producing a biliary tract cancer organoid model of biliary tract cancer by bringing the biliary tract cancer organoid of claim  10  into contact with a biliary tract cancer-inducing substance; and   a step of measuring viability of the organoid after bringing the test substance into contact with the biliary tract cancer organoid model of biliary tract cancer and comparing the measured viability with the viability of a biliary tract cancer organoid model of biliary tract cancer that is not brought into contact with the test substance.